Catalytic solid substrate room temperature phosphorimetry for the determination of trace rhamnose based on its condensation reaction with calcein

► A catalytic phosphorimetry was established for the determination of rhamnose (Rha). ► The detection limit is 7.8 zg spot−1, which was more sensitive than voltammetry. ► The results for determining Rha of this method were in accordance with those of HPLC. ► R-Rha was analyzed using HPLC, MS and NMR, and the mechanism of method was discussed. ► The research promoted the progress of phosphorimetry and human health protection.